Protein, an antibody and measurement of the protein

ABSTRACT

Methods for determining the occurrence or level of a mammalian protein in a sample and methods for determining the capability of a compound to transform or to inhibit the transformation of a selected mammalian protein in pro-phospholipase B form to an enzyme active phospholipase B form employ a pro-phospholipase B (PLB-II) mammalian protein which comprises at least one SU2 subunit comprising SEQ ID NO: 3 and having a molecular weight within the range of 30-60 kDa. Methods for determining the capability of a compound to enhance or inhibit the enzymatic activity of a selected protein having phospholipase B enzyme activity employ a protein comprising an activated form of such a pro-phospholipase B (PLB-II) mammalian protein.

TECHNICAL FIELD

The present invention relates to a novel kind of protein and itssubunits. The protein, in particular a subunit thereof, is enzymaticallyactive typically by exhibiting phospholipase B activity or by being apro-phospholipase B (i.e. are PLBs). The protein, its DNA and itsactivation route are different from the PLBs so far recognized. It hastherefore been named PLB-II (the human variant HPLB-II) in order todistinguish it from previously known PLBs. The human variant is relatedto human hypothetical protein F1122662 (SEQ ID NO: 1).

BACKGROUND ART

The neutrophil plays an important role in both innate immunity and ininflammatory reactions in human disease (Burg et al., Clin. Immunol. 99,7-17 (2001)). The neutrophil eliminates invading microorganisms throughphagocytosis, generation of reactive oxygen metabolites and release ofmicrobicidal substances stored in different granules in neutrophil.Apart from secretory vesicles, neutrophils contain azurophil, specific(secondary) and gelatinase-containing granules (tertiary) (Borregaard etal., Blood 89, 3503-3521 (1997)) formed in the bone marrow at subsequentstages of neutrophil maturation (Borregaard et al., Blood 85,No3,812-817 (1995)). During neutrophil-mediated inflammatory reactions thesecretory vesicles are mobilized first upon stimulation, followed by thetertiary, the secondary and the azurophil granules (Sengelov et al., J.Immunol. 154, 4157-4165 (1995); and Sengelöv et al. J. Immunol. 150No.4, 1535-1543 (1993)). Upon phagocytosis, the azurophil granules fusewith the phagosomes, which causes the release of proteolytic andbactericidal factors into the phagolysosome, where the invadingmicroorganism is killed and digested (Burg et al., (2001) cited above).

Phospholipase Bs (Ghannoum et al., Clin. Microbiol. Rev. 13, 122-43,Table (2000)) are a heterogeneous group of enzymes that can hydrolyzeboth the sn-1 and sn-2 fatty acids of glycerophospholipids, and thusdisplay both phospholipase A₁ or phospholipase A₂ and lysophospholipaseactivities. Several PLBs have been identified in various microorganisms(Ghannoum et al., (2000) cited above; and (Farr et al., J. Bacteriol.183, 6717-6720 (2001)), fungi (Ghannoum et al., (2000) cited above),Dictystelium discoideum (Ferber et al., Eur. J. Biochem. 14, 253-257(1970)) and in the brush border membrane of mature enterocytes fromguinea pig (Gassama-Diagne et al., J. Biol. Chem. 267, 13418-13424(1992)), rat (Tojo et al., J. Biol. Chem. 273, 2214-2221 (1998), rabbit(Boll et al., J. Biol. Chem. 268, 12901-12911 (1993)) and humanepidermis (Maury at al., Biochem. Biophys. Res. Commun. 295, 362-369(2002)). PLBs are also important components of venoms from bees andsnakes. Bacterial and fungal PLBs have been reported to be virulencefactors that damage host cells, while PLBs of enterocytes from mammalsare involved in digestion of dietary lipids, and PLB expressed in humanepidermis probably plays a role in the differentiation process and isinvolved in the epidermal barrier function.

The full length of the FLJ22662 protein comprises 552 amino acidresidues (SEQ ID NO: 1, AAH63561). A recombinantly produced FLJ22662protein containing a sequence of 223 residues from the full lengthvariant (a.a 330-552) (AAH00909, Strausberg et al., Proc. Natl. Acad.Sci. U.S.A. 99 (26), 16899-16903 (2002)) fused to GST is commerciallyavailable from Novus Biologicals (Littleton Colo., US). Suggested usesare Western blot, ELISA, and assay development. US patent applications20070059717, 20070004038, 20060252056, 20060240426, 20060199204,20060194199, 20060111314, 20060073496, 20060046259, 20060019256,20050208500, 20050095607, 20040076955, 20040005563, 20030170743, and20030165949 (issued as U.S. Pat. No. 7,189,507) are related to thediagnosis and therapy of various diseases and mention FLJ22662 proteinsand their genes as one out of many other proteins that might be ofinterest.

All US patents and patent applications including published Internationalpatent applications designating the US are hereby incorporated in theirentirety into the present specification.

OBJECTIVES

The identification and characterization of novel granule proteins inhuman neutrophils is an important approach to study the functions ofhuman neutrophils. In searching for novel granule proteins, we found aprotein of molecular weight 130 kDa in acid extracts of granulocytescontaining 25 and 45 kDa subunits/molecules. The amino acid analysisidentified this protein as a product of a gene (FLJ22662) which encodesa protein of 63 kDa with unknown function. Comparison of this proteinwith the GenBank sequence database using the BLAST program revealed anamino acid sequence similarity with Dictyostelium phospholipase B (PLB),suggesting a putative PLB. The objectives of the invention comprise:

-   -   1. Understanding of the biological significance in health and        disease of the novel protein.    -   2. Development of new drug principles based on the found        activity of the protein as such including its individual        subunits, e.g. PLB-II activity. This could be accomplished        by: a) using the protein as such or its individual subunits        either in native or in recombinant form, or b) mimicking the        activity of the novel protein or its subunits for the design of        new drug principles, or c) the development of principles that        facilitates the conversion of proforms into active forms.        -   This approach should prove valuable in the design of new            antibiotic drugs, but could also prove valuable as an            antithrombotic principle in other situations where the            activity is physiologically important.    -   3. Development of inhibitory principles for the activity, e.g.        PLB-II activity. This could be accomplished by a) development of        drugs that inhibit the activity, or b) drug principles that        prevent the transformation of pro-forms into active forms.        -   The inhibition of activity should prove valuable in a large            number of human diseases such as inflammatory disease,            cardiovascular disease, lipid disorders in which neutrophil            granulocytes and monocytes/macrophages are known to be            involved etc.    -   4. Development of sensitive assays for the measurement of the        activity of the protein and/or its individual subunits either        alone or in combination. The assay principles may be based        on: a) immunological principles including also other affinity        principles between forms of our novel protein and a        non-immunological affinity counterpart, b) the measurement of        the enzymic activity, and/or c) detection of mRNA expression of        the gene that relates to the protein (FLJ22662 in humans). This        objective includes diagnostic methods in which the level of the        novel protein, its individual subunits and/or mRNA expression in        a body fluid or other tissue is/are deviating from the        corresponding levels of healthy individuals and a used as a        marker for diseases and other physiological conditions that are        related to deviating levels of these biological entities.

THE INVENTION

The invention comprises three main aspects: 1) A mammalian protein thatcomprises one or more subunits each of which is related to a fragment ofhypothetical human protein FLJ22662 or to corresponding fragments in thehomologous hypothetical proteins from mammalian species other than Homosapiens. There may possibly also be other subunits in the protein of theinvention. 2) An antibody preparation specific for the mammalian proteindefined in this specification. 3. A method for measuring the occurrenceof an analyte in a sample. The analyte is related to the mammalianprotein of the first main aspect.

A Novel Mammalian Protein (First Main Aspect)

This aspect relates to a mammalian protein that is characterized inbeing derived from

-   -   a) hypothetical human single chain protein FLJ22662 which        exhibits the amino acid sequence of SEQ ID NO: 1 and in        gelfiltration and possibly also in vivo is present as a protein        containing four non-covalently associated single chain        polypeptide subunits (SU subunits) that pair-wise are different        non-overlapping fragments (two SU1 subunits and two SU2        subunits) of SEQ ID NO: 1, and/or    -   b) a homologous hypothetical protein of a mammalian species        other than Homo sapiens, which in a similar manner as FLJ22662        is capable of giving fragments that can associate with each        other to give a multimeric protein containing SU1 and SU2        subunits which are homologous to the subunits/fragments deriving        from the FLJ22662 protein.

The term “hypothetical protein” will in the context of the inventionmean a single chain protein that like FLJ22662 can be isolated frombiological material as a native multimeric protein containing at leasttwo non-covalently associated single chain polypeptide subunits (SU1 andSU2 subunits) that correspond to different non-overlapping fragments ofthe hypothetical protein. The in vivo occurrence of this kind ofhypothetical proteins is often too low or too rare to be measured and/ordetected. The sequence of the SU1 subunit is typically located closer tothe amino-terminal end of the hypothetical protein than the sequence ofthe SU2 subunit.

The mammalian protein of the invention is further characterized incomprising an SU1 subunit and/or an SU2 subunit. One or both of thedifferent subunits may be present in the protein as a duplicate, atriplicate etc. and/or there may be one or more additional subunits ofother kind(s). The SU1 subunits and/or the SU2 subunits may derive fromthe same or from different species (man-made forms). Individual SUsubunits may also be chemically derivatized forms and/or recombinantforms including mutated forms that do not exactly correspond to SUsubunits of any particular mammalian species.

The SU1 subunit comprises SEQ ID NO: 2 or a variant thereof which isobtained by substitution, deletion and/or addition of one or more aminoacid residues in SEQ ID NO: 2. The SU2 subunit comprises SEQ ID NO: 3 ora variant thereof which is obtained by substitution, deletion oraddition of one or more amino acid residues in SEQ ID NO: 3. The term“variant” includes that the sequence of an SU variant exhibit a sequenceidentity of ≧50%, such as ≧60% or ≧75% or ≧80% or ≧90% or ≧95% with thesubunit it is a variant of, i.e. SEQ ID NO: 2 or SEQ ID NO: 3. Theranges include that sequences/ subunits/fragments may derive fromhomologous proteins of different species as well as variants obtained bygenetic engineering. The term “sequence identity” refers to the programused in the experimental part to determine sequence identity. SEQ ID NO2 and SEQ ID NO 3 occur as non-overlapping regions in SEQ ID NO: 1 withSEQ ID NO: 2 being located closer to the amino-terminal end of SEQ IDNO: 1 than SEQ ID NO: 3.

The substitution, deletion and/or addition of residues shall preferablynot extinguish all important biological functions of native forms of thesubunits and/or the corresponding multimeric protein. Their capabilityof associating to each other or to exhibit enzyme activity, such asPLB-II or pro-PLB-II activities, should at least be partially retained,for instance.

In one variant a part of a specified length of the sequence in an SUsubunit is identical to a part of the same length A) in the sequence ofhypothetical FLJ22662 protein and/or B) in the sequence of acorresponding homologous hypothetical protein of a mammalian speciesother than Homo sapiens.

-   A) In a subvariant an SU1 subunit and/or an SU2 subunit comprise at    least one, two or more amino acid sequences of ≧5, such as ≧10 or    ≧15≧25 or ≧50 amino acid residues which sequences are present in SEQ    ID NO: 1 or in corresponding parts of the sequence of a homologous    hypothetical single chain protein of a mammalian species other than    Homo sapiens.-   B) In another subvariant,    -   a) an SU1 subunit comprises at least one, two or more amino acid        sequences of ≧5, such as ≧10 or ≧15≧25 or ≧50 amino acid        residues which sequences are present in SEQ ID NO: 2 or in        corresponding parts of the sequence of a homologous hypothetical        single chain protein of a mammalian species other than Homo        sapiens, and/or    -   b) an SU2 subunit comprises at least one, two or more amino acid        sequences of ≧5, such as ≧10 or ≧15≧25 or ≧50 amino acid        residues which sequences are present in SEQ ID NO: 3 or in        corresponding parts of the sequence of a homologous hypothetical        single chain protein of a mammalian species other than Homo        sapiens.

This kind of part sequence in an SU1 subunit should be non-retrievablein an SU2 subunit and vice versa for an SU2 subunit.

The molecular weight M_(w) of the mammalian protein of the inventiondepends on the number and kinds of SU subunits in it as well as on thespecies origin of the subunits and on particular deletions,substitutions or additions of amino acid residues. Typical M_(w)s arewithin the range of 15-240 kDa presuming the number of subunits is 1-4.For single SU1 subunits the typical range is 15-35 kDa and for singleSU2 subunits 30-60 kDa. For variants that comprise four subunits thetypical range is 60-240 kDa with a more narrow range being applicable tovariants in which there are two SU1 and two SU2 subunits, e.g. 90-190kDa, such as 110-150 kDa. The M_(w)s of pure human variants arepresented in the “Experimental part”. These M_(w) ranges primarily applyto the polypeptide chain(s) of the protein and subunits of theinvention. They do not include the M_(w) of groups that have been addedby recombinant fusion with other proteins or by chemical derivation toattach other large molecular weight entities. The ranges given above inparticular refer to M_(w)s obtained by gel filtration for multimericproteins and by SDS-PAGE under denaturing conditions for individualsubunits.

In preferred variants the protein of the invention is a lipase, such asphospholipase and most typical a phospholipase B. This includes that theprotein is an active enzyme or is a pro-enzyme that is possible totransform/activate to an active enzyme, e.g. to a phospholipase B. Inorder for the protein to be a pro-enzyme or an active enzyme (PLB-II),it seems imperative that the novel protein has at least an SU2 subunitand optionally is devoid of the SU1 subunit. It is likely that some kindof degradation of the SU2 subunit is necessary for enzyme activity, forinstance by cleaving off parts corresponding to a reduction in molecularweight of the SU2 subunit. The reduction in M_(w) for activation istypically ≧0.5 kDa, such as >1 kDa or ≧2 kDa or ≧3 kDa but presumed tobe ≦10 kDa. Alternatively the reduction in M_(w) may be measured inpercentage, e.g. ≧1%, such as ≧2% or ≧4% or ≧6% but presumed to be ≦20%of the Mw of the subunit concerned, e.g. an SU2 subunit. Based onresults so far obtained the reduction is typically ≦5 kDa or ≦10% forhuman variants and the like.

The amino acid sequence of the various SU subunits are lacking thelipase consensus sequence, i.e. a sequence of five amino acid residuesstarting and ending with glycine and having serine in the middle andindependently any kind of amino acid residues at the two remainingpositions (Schrag et al., Methods Enzymol. 284:85-107, 85-107 (1997)).

The mammalian protein of the invention may be in derivatized form wherethree important variants are 1) labelled forms, 2) immobilized orimmobilizable forms, and 3) fused forms, i.e. recombinant forms in whichone or more other compounds exhibiting polypeptide structure has beenfused to one or more subunits of the novel protein.

The mammalian protein in labelled form is typically a conjugate betweenthe protein and an analytically detectable group. Detectablegroups/labels are mainly of two kinds 1) signal-generating labels, and2) affinity labels. Typical examples of the first kind of labels arefluorophors, luminophors, such as bioluminophors and chemiluminophors,radioactive groups, enzymatically active groups, such as enzymes assuch, substrates, co-substrates, inhibitors, enhancers, promoters,cofactors, coenzymes etc., particles, e.g. metal particles such as goldparticles, etc. Typical examples of the second kind of labels are amember in a bioaffinity pair, such as biotin andavidin/streptavidin/neutravidin etc., hapten/antigen andanti-hapten/antigen antibodies, complementary nucleic acids, constantregion of an antibody and anti-constant region antibody (naturallyoccurring conjugate) or microbial Ig binding proteins (protein A, Getc.) etc. The techniques for producing these kinds of conjugates arewell-known in the field and typically involve chemical coupling of thelabel to the protein of the novel mammalian protein, or, as analternative if the label exhibits polypeptide structure, recombinantfusion of the label to the appropriate subunit(s) of the mammalianprotein of the invention.

In immobilized or immobilizable forms the mammalian protein is attachedto a solid phase or is capable of being attached to such a phase byexhibiting a suitable functional group that matches a “counter-group” onthe solid phase.

Solid phases are well known in the field and encompass surfaces, such asinner surfaces of inner walls of reaction vessels, particles, forinstance in the form of beads, which may be porous or non-porous, porousmonolithic plugs, membranes, sheets etc. Beads may be in suspended form(expanded beds, stirred suspension etc.) or in the form of packedbeds/sedimented beds. The material in the solid phase, e.g. inparticles, is typically polymeric, for instance a synthetic polymer or abiopolymer and includes also inorganic polymers such as glasses. Theterm biopolymer includes semi-synthetic polymers comprising a polymerchain derived from a native biopolymer. The particles and other forms ofsolid phases (e.g. particles packed to a bed) are typically hydrophilicin the sense that they will be saturated by water by the action ofcapillarity (self-suction) if in contact with an excess of water. Theterm also indicates that the surfaces of the solid phase material shallexpose a plurality of polar functional groups each of which comprises aheteroatom selected amongst oxygen, sulphur, and nitrogen. Appropriatefunctional groups can be selected amongst hydroxy groups, straightethylene oxide groups ([—CH₂CH₂O—]_(n), where n is an integer >0), aminogroups, carboxy groups, sulphone groups etc., for instance, withpreference for those groups that are uncharged independent of pH.Hydrophobic solid phase materials, e.g. in the form of particles, may behydrophilized, typically by introducing hydrophilic groups on theirsurface, for instance by coating.

The techniques for immobilization may be selected amongst those that areknown in the field, for instance via covalent bonds, affinity bonds (forinstance biospecific affinity bonds), physical adsorption (mainlyhydrophobic interaction) etc. Examples of biospecific affinity bondsthat can be used are bonds between avidin/streptavidin/neutravidin etc.and a biotinylated affinity reactant, high affinity antibody and ahaptenylated affinity reactant etc.

The mammalian protein of the present invention is in isolated form(=enriched form). This typically means that the protein is present in apurity that corresponds to an enrichment relative to its concentrationin an acidic extract of granules of granulocytes of the same speciesorigin as the protein, and in particular relative to the totalconcentration of proteins in such an extract. The acidic extract is ofthe same kind as the one used as starting material for our purificationof the innovative protein. See the experimental part. Typical enrichmentfactors are ≧10, such ≧10² or ≧10³ or ≧10⁴.

The protein may be present in compositions that are in liquid or dryform, such as in spray-dried, air-dried or lyophilized form. Thecompositions may contain buffers and/or stabilizing agents selected fromvarious compounds exhibiting carbohydrate structure or other polyhydroxystructures, e.g. selected among sugar alcohols, mono- and disaccharides,oligosaccharides, polysaccharides. Specific examples are glucose,sacharose, lactose, trehalose, dextran etc. Potentially interestingsubstances to be incorporated into compositions that are to be handledin dry form are substances that in dried form are capable of existing ina glassy state.

The mammalian protein of the invention can be obtained from nativematerial by working up acid extracts obtained from granules ofgranulocytes. See the experimental part. In the future it is likely andprobably will also be preferred to obtain at least the polypeptidechains of the subunits of the mammalian protein of the invention byfirst producing recombinantly the hypothetical single chain protein,such as FLJ22662, followed by enzymatic fragmentation, possibly bycarrying out the appropriate folding and/or subunit association prior orsubsequent to the fragmentation. Alternatively each kind of SU subunitsis produced separately whereafter folding and subunit association areallowed to proceed. Still another alternative is to employ solid phasesynthesis for the different subunits which subsequently are folded andassociated to form a multimeric form of the innovative protein.

Antibodies Specific For The Protein of the Invention (Second MainAspect).

This aspect encompasses an antibody preparation (=composition) which isspecifically directed against a mammalian protein as defined for thefirst aspect. The specificity is for one or more different epitopes onthe mammalian protein that are unique for the protein. The term “unique”in this context implicates that the epitopes are not occurringdisturbingly in other molecules that are present together with themammalian protein, for instance in vivo, or when the antibodypreparation is to be used in assaying methods comprising complexformation between the mammalian protein and an affinity counter part asdiscussed for the third aspect of the invention.

The antibody preparation may be specific for one or more epitopes on a)an SU1 subunit or b) an SU2 subunit or c) a multimeric form of the novelprotein. Alternative c) encompasses specificity for epitopes that areunique for the multimeric form and/or for SU1 and/or SU2 epitopes thatare exposed on the multimeric form.

The antibody preparation of the invention is polyclonal or monoclonaland may include a mixture of different monoclonals, e.g. two or more andtypically ≦10.

The term “antibody” includes full length antibodies, antigen-bindingfragments and chemical derivatives and various recombinantly producedforms, such as single-chain antibodies, fused forms, chimeric forms etc.In its most general meaning the term also encompasses any man-madeconstruct that can be obtained in a form that exhibits specific affinitytowards a protein of the invention, e.g. antibodies. Individual antibodyactive entities (e.g. capable of binding to the epitopes discussedabove) in the antibody preparations of the invention may be derivatizedin the same manner as outlined for the mammalian protein of theinvention, e.g. the same kinds of labels and solid phases as discussedfor the first main aspect may be attached to the entities.

Methods Involving Measurement of the Mammalian Protein (Third MainAspect)

This aspect is a method for determining the occurrence or level of ananalyte in a sample. The characteristic feature is that the analyte isrelated to the mammalian protein defined in this specification. Theanalyte may thus be a) the mammalian protein as such, b) an inhibitor orenhancer of the activity of the mammalian protein, c) an inhibitor orenhancer of the transformation of a proform of an enzyme active form ofthe mammalian protein to an active form exhibiting at least a higheractivity than the proform, etc. Alternative c) that the enhancer may bean enzyme that promotes the conversion into the active form of themammalian protein or an enhancer for such an enzyme. Alternative b) andc) include also determining the capability of a particular compound tobe an enhancer or an inhibitor. The determination of occurrence or levelof analytes according to alternatives a)-c) typically involvesmeasurement of the enzymatic activity of the mammalian protein of thefirst aspect.

The terms “occurrence” and “level” comprise concentrations(quantitative), e.g. amount per unit volume, or relative amounts, suchas relative to a reference substance that may be internal (i.e. ispresent together with the analyte in the sample) or external (i.e. isseparate from the sample). An internal reference substance is typicallyadded separately to the sample or the biological fluid, or is presentalready in the original biological material from which the analytederives. The terms also include biological activity, for instance enzymeactivity, if the compound is an enzyme active form of the mammalianprotein as defined herein, for instance exhibiting phospholipase Bactivity and/or pro-phoshoplipase B activity. The terms also includepresence or absence of the analyte (i.e. qualitative measurements).

The sample may be any kind of sample that may contain the mammalianprotein and/or an entity related to the protein such as enhancer or aninhibitor of biological activity of the protein (see above). The sampletypically derives from a biological fluid. Typical biological fluidsencompass cell culture supernatants, tissue and tissue homogenates,blood and various blood fractions such as serum or plasma, lachrymalfluid, regurgitated fluid, urine, sweat, semen, cerebrospinal fluid,gastric juice, saliva, lymph, lung lavage fluid, intestinal fluid etc.as well as various other liquid preparations containing a bio-organiccompound, for instance various liquids obtained in various steps duringthe purification of the mammalian protein of the invention from itsnative sources (see the experimental part) or from its recombinantproduction.

In variants in which the capability of a particular compound to act asan inhibitor or enhancer is determined, the sample is typically thereaction mixture in which the compound is to interact with the mammalianprotein of the invention. The sample is typically aqueous. It may be aliquid possibly containing insoluble material, or some kind of solidmaterial, such as tissue, a gel, nitrocellulose sheet etc., that maycontain a liquid part.

There are thus at least two main kinds of biological fluids: A) nativebiological fluids of a mammalian individual including also fluidsderived from the native fluid by sample preparation, such as dilutions,fractions (plasma and serum from whole blood) etc., or B) processliquids obtained during production and/or isolation and/or enrichment ofthe mammalian protein from mammalian individuals or from recombinantprocesses generating the protein. If a sample is derived from amammalian individual, the sample is typically taken for diagnosticpurposes.

The terms “diagnosis”, “diagnostic purposes” and the like in the contextof the invention encompass the initial diagnostication of diseasedconditions as well as the follow-up or monitoring of the diseasedconditions after an actual diagnosis has been established, e.g. during atreatment or healing process or other kinds of observation periods (e.g.before the outbreak of the disease).

A first subaspect is a method for diagnosing a diseased conditionassociated with an abnormal level of the mammalian protein of theinvention (analyte) in mammalian individuals (patients). The protein istypically a phospholipase B (PLB-II) or a pro-phospholipase B(pro-PLB-II). This subaspect comprises the steps of:

-   -   (i) measuring the level of the mammalian protein in a sample        which is derived from a biological fluid originating from a        mammalian individual,    -   (ii) taking a found level that deviates from the level for        normal (healthy) individuals of the same species, e.g. by being        elevated, as an indication of said individual suffering from the        diseased condition.

The level may be increased or decreased. Diseased conditions of interesttypically relate to the biological activity of the mammalian protein ofthe invention, for instance its phospholipase activity or the ability ofthe subunits (SU1, SU2 etc.) to exist as a multimeric protein or infully or partly dissociated form. Increased levels are likely to beindicative of diseased conditions, such as i) an inflammatorydisease/inflammation, ii) a microbial infection, such as a viral,bacterial or prion infection, iii) a cardiovascular disease, or iv) alipid disorder, for instance involving neutrophil granulocytes and/ormonocytes/macrophages.

Step (i) of the first subaspect may utilize

-   -   Alternative (a): one or more affinity counterparts specific for        a native form of the mammalian protein described herein, for        instance an antibody as defined for the second main aspect in an        assay format as described for the second subaspect below, or    -   Alternative (b): enzymic reactants, such as substrate,        cofactors, enzyme activators etc., i.e. the method is an enzyme        assay.

Other alternatives encompass chromatographic procedures that results infractions in which the mammalian protein of the invention is enrichedand thereafter measured.

A second subaspect is a biospecific affinity assay that utilizes anantibody preparation according to the second main aspect. This subaspectcomprises the step of:

-   -   (i) contacting the sample with the antibody preparation specific        for the mammalian protein described herein under conditions        permitting formation of a complex between the antibody and the        mammalian protein in an amount that is related to the level of        the mammalian protein in the sample,    -   (ii) measuring the level of the complex formed in step (i)        and/or the level of the antibody not complexed to the protein,        including the presence or absence of said complex, and    -   (iii) calculating the level of the mammalian protein in the        sample and/or in the biological fluid from which the sample        derives from the level measured in step (ii).

The biological fluid is typically one of the main kinds discussed above(A and B).

If the sample and the biological fluid derive from a mammalianindividual, the second subaspect may be performed for diagnosticpurposes and will then comprise an extra step (iv) that will be the sameas step (ii) of the first subaspect. In other words the second subaspectwill then coincide with alternative (a) of the first subaspect.

The particular assay format to be used in the second subaspect isselected amongst those that are available for biospecific affinityassays. These formats as well as the principles for selection are wellknown in the field. They encompass that one or more affinitycounterparts, for instance one or more antibody preparations specificfor the analyte are used for the formation of an affinity complex, thelevel of which then is measured and related to the level of the analytein the sample. The conditions are selected such that the level of thecomplex becomes a function of the level of analyte in the sample and inthe biological fluid from which the sample derives. The formats may ormay not use an affinity reactant that exhibits a label of the kindsdiscussed above for labelled forms of the mammalian protein of theinvention (see first aspect of the invention), e.g. a labelled form ofa) a mammalian protein of the first main aspect, or b) an antibodyspecific for this protein. During an assay, a labelled affinity reactantis typically incorporated into an affinity complex in an amount that isa function of a) the level of analyte in the sample, and/or b) the levelof the complex formed that contains the mammalian protein and anantibody specific for the protein. The formats may or may not use anaffinity reactant that is immobilized or immobilizable to a solid phaseas described for the protein of the first main aspect. One way ofgrouping formats utilizing labelled reactants and/or immobilized orimmobilizable reactants is into competitive and non-competitive assays.The sandwich format is a typical non-competitive format and utilizes asa rule at least two affinity counterparts that are specific for themammalian protein (analyte) so that they simultaneously can bind to theanalyte. One of these counterparts typically exhibits a label while theothers exhibits another label or is immobilized or immobilizable to asolid phase. The competitive formats typically utilize an analyteanalogue, i.e. the mammalian protein in labelled form or in immobilizedor immobilizable form. The analogue is typically competing with theanalyte for binding to a common affinity counterpart (e.g. antibody)that is in limiting amount. The affinity counterpart may be in labelledform in the case the analyte analogue is in immobilized or immobilizableform and in immobilized or immobilizable form if the analyte analogue isin labelled form. So called displacement assays are often considered ascompetitive formats. The formats may also be divided into heterogeneousand homogeneous formats where heterogeneous formats require a separationof labelled reactant incorporated in a complex from the same labelledreactant not incorporated in the complex before the measuring in step(ii) is carried out. The homogeneous formats do not require this kind ofseparation. Biospecific assay formats also include immunoblotting,agglutination formats (particles as labels), nephelometric/turbidometricformats etc.

The third subaspect comprises a) that the mammalian protein is enzymeactive, for instance that it exhibits phospholipase B (PLB-II) activityor pro-phospholipase B (pro-PLB-II) activity, and b) that the activitylevel of either one or both of these entities is measured.

A first variant, the third subaspect comprises the steps of

-   -   (i) incubating the sample containing the mammalian protein of        the type described in the first main aspect with the appropriate        substrate and other components necessary for substrate        conversion,    -   (ii) measuring substrate conversion as a function of time, and    -   (iii) calculating the enzyme activity level of the mammalian        protein in the sample and/or the biological fluid from which the        sample derives based on the substrate conversion rate measured        in step (ii).

It is important to secure that the sample is devoid of other enzymesthat may cleave one or more of the acyl ester bonds (sn-1 and sn-2) thatour novel phospholipase B does, for instance phospholipase A1,phospholipase A2, and other phospholipase Bs etc. This can beaccomplished by removing such enzyme activities prior to step (i) or ifthe sample by definition is lacking such activities.

The sample typically derives from one of the two main kinds ofbiological fluids discussed above (A and B). If derived from a mammalianindividual, the method may be performed for diagnostic purposes and themethod carried out as part of the diagnostication of a diseasedcondition of the individual. The variant will then comprise a fourthstep (iv) that will be the same as step (ii) in the first subaspect. Inother word this variant then coincides with alternative (b) of the firstsubaspect.

A second variant of the third subaspect is a test for the capability ofa compound (analyte) to promote or inhibit the transformation of apro-phospholipase B (pro-PLB-II) of the invention to a phospholipase B(PLB-II) of the invention. In this variant the compound to be tested isthe analyte and included in the incubation mixture of step (i). Thisvariant thus comprises the steps of:

-   -   (i) bringing the pro-phospholipase B, a substrate for        phospholipase B and the compound to be tested in contact with        each other under activating conditions,    -   (ii) measuring conversion of the substrate as a function of        time, and    -   (iii) determining from said conversion the capability of the        test compound to promote or inhibit the activation of        pro-phospholipase B to phospholipase B.

A third variant of the third subaspect is a method for testing thecapability of a compound to inhibit or enhance the activity of aphospholipase B of the invention. In this variant the compound to betested is the analyte and included into the incubation mixture of step(i). This third variant thus comprises the steps of:

-   -   (i) contacting phospholipase B of the invention with the        compound to be tested and a substrate for phospholipase B with        each other under conditions that allow substrate conversion,    -   (ii) measuring conversion of the substrate as a function of        time, and    -   (iii) determining the enhancing or inhibiting capability of said        compound from a comparison between the measured conversion in        step (ii) and the conversion rate for selected standard        conditions.

Both the second and the third variants of the third subaspect may bepart of a drug development process in which the final drug is to be usedfor the treatment of a diseased condition that is related to nativelyoccurring forms of a mammalian protein of the first aspect of theinvention.

BEST MODES OF THE INVENTION

The best modes are:

1) Protein aspect: The human variants that are isolated andcharacterized in the experimental part. It is believed that in thefuture recombinant produced variants will become more important andtherefore preferred. 2) Antibody aspect: The polyclonal antibodypreparations used in the experimental part, in particular ofspecificities making them useful for biospecific assays of native formsof proteins of the first aspect in samples derived from biologicalfluids from humans. Important future variants will typically bemonoclonal. 3) Method aspect: Biospecific assays for the native forms ofthe proteins defined in the first aspect and being present in samplesderived from biological fluids of humans. Important future assays willtypically be in the sandwich format and/or utilizing one or moreantibody preparations that are monoclonal.

Experimental Part

Methods

Preparation of granule proteins. Granules were isolated from the buffycoat of normal human blood by a modification of the method describedearlier (Peterson et al., Eur. J. Haematol. 40, 415-423 (1988)). Thebuffy coats, approximately 51 originating from 100 healthy blood donors,were mixed with an equal volume of 2% Dextran T-500 inPhosphate-buffered saline (Dulbecco, without calcium and magnesium). Thegranulocyte-rich plasma was collected after sedimentation of the redcells for 1 h at room temperature. The granulocytes were washed twice inPBS and once in 0.34 M sucrose by centrifugation at 400 g for 10 min.The granulocyte pellet was resuspended in 5 volumes of 0.34 M sucrose.Isolated cells were then disrupted by nitrogen cavitation. Cellsuspension was mixed with an equal volume of 0.34 M sucrose and thecells were pressurized at 4° C. for 30 mM under nitrogen at 52 bar withconstant stirring in a nitrogen bomb (Parr Instrument Company, Moline,Ill.). The cavitate was then collected into an equal volume of 0.34 Msucrose, 0.3 M NaCl and centrifuged for 20 mM at 450 g at 4° C. Thesupernatant was centrifuged for 20 min at 10000 g at 4° C. to sedimentthe granules. After one cycle of freezing and thawing the granules wereextracted with 5 volumes of 50 mM acetic acid for 1 h at 4° C. An equalvolume of 0.4 M sodium acetate pH 4.0 was added and the extractionprocedure was continued with magnetic stirring for 4 h at 4° C. Thegranule extract was then concentrated to approximately 5 ml using YM-2filter (Amicon Corporation, Lexington, USA).

Chromatographic procedures. The procedure comprised four distinct steps(A-D) on three different columns:

-   -   Step A gel filtration: Acid extracts of granules obtained from        human granulocytes were loaded on a Sephadex G-75 column        (2.5×90 cm) and eluted by 0.2 M NaAc pH 4.5. The majority of the        protein of the invention was contained in the second peak        (elution volume/fractions 58-69 ml), as judged by SDS-PAGE after        further separation of proteins in each pool on Mono-S column.    -   Step B ion-exchange chromatography: FPLC-system (Amersham        Biosciences, Uppsala, Sweden) with a strong cationic exchanger        Mono-S prepacked column Fractions/elution volumes of 58-69 ml        from the gel filtration chromatography were applied to the        Mono-S column pre-equilibrated with 0.1 M NaAc pH 4.0 and eluted        by a linear gradient from 0 to 1.0 M NaCl in 0.1 M NaAc pH 4.0        (0.1-0.5 M NaCl for elution volume 6-27 ml and finally ending at        0.5 ml at elution volume 36 ml). The protein of the invention        was eluted in the fractions/elution volume 19-22 ml (the second        peak).    -   Step C ion exchange chromatography: The same system and column        as in step B but the column was now equilibrated with 0.006 M        sodium phosphate pH 7.4 and eluted by a linear gradient from        0.006 to 0.5 M sodium phosphate pH 7.4 for elution volumes        5-24 ml. The protein of the invention was eluted in the main        second peak (fractions/elution volume 11-14 ml).    -   Step D hydroxy apatite chromatography: The fractions containing        the protein of the invention from step C were applied to a        hydroxyapatite column (BioRad) equilibrated with 0.02 M sodium        phosphate buffer pH 7.2 and eluted with a linear gradient from        0.02 M sodium phosphate buffer pH 7.2 to 0.4 M sodium phosphate        pH 6.8 for fractions/elution volumes 5-25 ml. Fractions/elution        volumes 19-22 ml contained pure protein.

Approximately 0.5-20 μg of proteins from steps one to four of thepurification were applied to SDS-PAGE, and proteins were visualized bysilver staining.

The proteinase inhibitors, phenylmethylsulfonyl fluoride (PMSF) (100mg/l) and Soybean trypsin inhibitor (SBTI) (100 mg/l) were added to allbuffers from the cell disruption step to the first ion-exchangechromatography. Proteins in the chromatograms were measured by theirabsorbance at 280 nm. Ultrafiltration of pooled fractions was performedon a YM-10 filter. Buffer change was performed on PD-10 columns(Amersham Biosciences, Uppsala, Sweden).

Electrophoretic analysis. Proteins in aliquots of 0.5-20 μg wereanalyzed with sodium dodecylsulfate-polyacrylamide gel electrophoresis(SDS-PAGE) under reducing and non-reducing conditions using precastNuPAGE gel (Novex, Carlsbad, Calif.), according to manufacturer'sinstructions. Proteins were visualized by silver staining.

Amino Acid Analysis.

In-gel digestion and extraction. The 25 kDa and 45 kDa bands from onelane in a Coomassie stained gel were excised and minced into smallpieces. The gel pieces were washed with distilled water, dehydrated withacetonitrile, and dried under vacuum. The sample was rehydrated anddigested with a digestion buffer containing 50 mM NH₄CO₃, 5 mM CaCl₂ and12.5 μg/ml of sequencing-grade modified trypsin (Promega, Madison, Wis.)over night at 37° C. The supernatant was collected, and the peptideswere extracted from the gel pieces with 20 μl of 25 mM NH₄HCO₃ andseveral times with 20 μl of 50% acetonitrile/5% formic acid.Supernatants from each extraction were combined.

MALDI Mass Spectrometry. The tryptic digest was analysed by MALDI-T of(Kompact MALDI 4, Kratos, UK). The mass analyser was scanned over a massto charge ratio (m/z) range of 500 to 4000 amu and the resulting spectrawere used for search of matching proteins in the NCBI database using theMascot search program (Matrix Science).

Nanoelectrospray Mass Spectrometry (MS/MS). After the initial peptidescanning, one peptide was subjected to MS/MS analysis (Micro Q-Tof,Manchester, UK) followed by search with the fragmentation spectra in theNCBI data using search program Mascot (Matrix Science).

Protein determination. Protein concentration was determined with aBio-Rad protein assay kit using bovine serum albumin as a standard byfollowing the manufacturer's protocol.

Enzyme assay. The reaction mixture (40 μl final volume) contained 10 mMsubstrates, 100 mM phosphate buffer containing 3 mM of sodium azide(NaN₃) and 0.5% Triton X-100, pH 7.4, and 0.5 μg of enzyme or asindicated. Since the formation of the product (fatty acid) was linearwith time for at least 24 h under the standard conditions, the reactionmixture was incubated for 18-20 h and the reaction was stopped bycooling on ice. Free fatty acid (FFA) was determined by means of theNEFA-C kit (WAKO chemicals, Neuss, Germany) according to theinstructions of the manufacturer.

Positional specificity of the purified enzyme was determined using1-palmitoyl-2-hydroxyl-phosphatidylcholine (1-palmitoyl-2-hydroxyl-PC)and 1-palmitoyl-2-[1-¹⁴C]palmitoyl-phosphatidylcholine(1-palmitoyl-2-[1-¹⁴C]palmitoyl-PC) as substrates. The hydrolyzingactivity of the enzyme at the position of sn-1 acyl ester bonds ofglycerophospholipids was determined as described above using1-palmitoyl-2-hydroxyl-PC as substrate. For the hydrolyzing activity ofthe enzyme at the position of sn-2, Dipalmitoylphosphatidylcholine(Dipalmitoyl-PC (50 nm/reaction) was mixed with radiolabeled PC(1-palmitoyl-2-[1-¹⁴C]palmitoyl-PC, 1×10⁵ cpm/reaction). The mixture wasdried out under nitrogen gas and resuspended in reaction buffer of 0.1 Msodium phosphate, 3 mM NaN₃ and 0.5% Triton X-100 at pH 7.4 bysonication to form micelles of phospholipids. The incubation was carriedout at 37° C. for 20 h, the reaction was stopped by mixing with 0.8 mlDole's reagent (32% isopropyl alcohol/67% heptane/1% 1M H₂SO₄, 20:5:1)and vortexed. After centrifugation 2 min at 1000 g, the upper phasecontaining free fatty acids was further purified by extraction with 50mg silica gel suspended in heptane. Radiolabeled fatty acids werequantified by scintillation counting.

Experiment A: Didecanoyl-PC was incubated with the purified protein atdifferent time of storage and free fatty acid release was measured.Enzymatic reactions were carried out with 0.5 μg of the purified proteinfor 20 h at 37° C.

Experiment B: Free fatty acid release from phospholipids, didecanoyl-PC(Dideca-PC), dipalmitoyl-PC (Dipalmi-PC), phosphatidylinositol (PI),phosphatidylethanolamine (PE) and lysophosphatidylcholine (Lyso-PC) wasmeasured. Enzymatic reactions were carried out with 0.5 μg of thepurified protein (stored at 4° C. for 15 w) for 18-20 h at 37° C.

Experiment C: The purified protein (stored at 4° C. for 16 w) waspreincubated at room temperature or 37° C. for 15 min with releasedmaterials (0.5 μg) induced from neutrophils by PMA before incubationwith Didecanoyl-PC. Enzymatic reactions were carried out with 0.5 μg ofthe protein for 20 h at 37° C. and free fatty acid release was measured.

Experiment D: Detection of phopholipase A₂ activity. Radioactivephospholipid, 1-palmitoyl-2-[1-¹⁴C]palmitoyl-PC was incubated without(Control) or with 1 μg of the purified protein (stored at 4° C. 16 w)for 20 h at 37° C. and radioactivity was counted as described in thematerials and methods section.

Analyses of the pH optimum, K_(m) and V_(max) The purified protein (0.5μg) was added to tubes containing Didecanoyl-PC at varying pH (4.0-9.0).The Km and V_(max)were calculated from Hanes plots of s/vi onDidecanoyl-PC concentration(s).

Preparative electrophoresis. Preparative gel electrophoresis wasperformed in the PrepCell system (Bio-Rad), following the instructionsof the supplier. The acrylamide concentration of the cylindricalseparation gel was 10%, and the gel was about 6 cm long. The stackinggel had an acrylamide concentration of 4% and was 2.5 cm long.

Antibody production. Laying hens were immunized with the purifiedprotein. For the immunization 0.5 ml antigens in phosphate-bufferedsaline (PBS) were emulsified with an equal volume of Freund's adjuvant.The first immunization was performed with Freund's complete adjuvant andthe booster immunization was with Freund's incomplete adjuvant. Theamounts of antigen used for each immunization were 5 μg. White Leghornhens were immunized intramuscularly in the breast muscle with theemulsified antigens. After the initial immunization, the animalsreceived booster injections with 2-week intervals for three times andeggs were collected continuously after the initial immunization periodof six weeks. Egg-yolk (2 ml) from individual eggs was mixed with 4 mlof 0.9% (w/v) NaCl, 5.25% (w/v) PEG 6000, 0.2% (w/v) NaN₃. Afterincubation overnight at 4° C., the mixture was centrifuged at 2000 g for10 min. The clear supernatant was used for the detection of antibodyresponse.

Cell Separation and Post Nuclear Supernatant Preparation

Blood cells were separated by density gradient centrifugation overisotonic 67% (v/v) of Percoll (Amersham Biosciences, Uppsala, Sweden).The interphase, containing the mononuclear cells and lymphocytes, wasremoved. The pellet fraction, containing erythrocytes and granulocytes,was treated for 15 min with ice-cold isotonic NH₄Cl solution (155 mMNH₄Cl, 10 mM KHCO₃, 0.1 mM EDTA, pH 7.4) to lyse the erythrocytes,followed by hypotonic lysis of residual erythrocytes. The remaininggranulocytes were washed twice in PBS (without Ca²⁺). To furtherseparate neutrophils from eosinophils, the isolated granulocytes wereincubated for 1 h at 4° C. with anti-CD 16 mAb-coated magneticmicrobeads (at a proportion of 1×10⁷ granulocytes in 30 μl PBS with 2%(v/v) newborn calf serum to 15 μl microbeads, Miltenyi Biotec, BergischGladbach, Gemany). The cells were subsequently allowed to pass through asteel matrix column in a magnetic field. Thereafter, the eosinophilsthat passed through were collected. The purity and viability of theeosinophils were >96 and 99%, respectively. After removing the magneticfield, the neutrophils were eluted with PBS. The purity of theneutrophils was more than 98%. Isolated eosinophils and neutrophils wereresuspended respectively in 6% (w/v) of sucrose solution containing 10μl/ml of protease inhibitor cocktail (Roche Diagnostics, Mannheim,Germany). Ultrasonication was performed to disrupt the eosinophils andneutrophils. Ultrasonicates were adjusted to 9% (w/v) of sucrose beforecentrifugation at 450 g for 20 min to eliminate the nuclei and intactcells. The post nuclear supernatants (25 μg) were loaded onto SDS-PAGEgels for immunoblotting. To obtain released materials, isolatedneutrophils were resuspended in Hanks balanced salt solution at around1×10⁸ cells /ml and stimulated with PMA (4×10⁻⁷ M) for 20 min at 37° C.After centrifugation the released material was aspirated. Under thiscondition, about 6% and 60% of primary and secondary granules werereleased from activated neutrophils, judged by the measurement ofmyeloperoxidase and human neutrophil lipocalin releases.

Immunoblotting. SDS-PAGE was performed under non-reducing conditionsusing precast NuPAGE gels (Novex, Calif.), according to manufacturer'sinstructions. For the immunoblotting, the proteins on the NuPAGE gelwere transferred to a nitrocellulose membrane (0.2 μm), as described inthe manufacturer's instructions. Additional binding sites were blockedby incubation of the nitrocellulose blot in 2% skim milk in 20 mMTris-HCl, pH 7.4 for 1 h. The blot was incubated overnight with chickenantibodies against the fragment of 45 kDa diluted 1/1000, followed by a2 h incubation with peroxidase-conjugated rabbit anti-chicken Ig Y(Immuno-System, Uppsala). Color was developed with Immuno-Blotcolorimetric assay kits (Bio-Rad, USA).

Results

Purification of a Protein of the Invention (Putative PhospholipaseB=Putative PLB)

Approximately 0.5-20 μg of proteins from steps one to four of thepurification were applied to SDS-PAGE, and proteins were visualized bysilver staining. The purified protein from step four of the purificationshowed only two bands at molecular weights of 25 kDa and 45 kDa underboth non-reducing and reducing conditions. However, these two moleculescould not be separated by chromatographic means including Mono-P and thereversed phase chromatography. On gel filtration chromatography thepurified native protein was eluted in one peak at a molecular weight ofaround 130 kDa, and on Mono-P chromatography the protein was eluted inone peak at a pH around 8.6.

Amino acid analyses. In order to identify the protein, the respectivebands at 25 and 45 kDa on the SDS-PAGE were digested by trypsin,followed by MALDI-T of and MS/MS analyses. The resulting spectrum wasused to search for matching proteins in the NCBI database, using theMascot search program. The search with the resulting spectrum from thebands at 25 kDa and 45 kDa yielded top scores of 76 and 116,respectively, for the hypothetical protein FLJ22662 with unknownfunction (a full-length protein of 63 kDa) (Protein scores greater than67 are significant, P<0.05). The identified amino acid residues byMALDI-T of and MS/MS are shown in Table 1. The residues from the 25 kDaband were found towards the N-terminus of the full length protein, whilethe residues from the 45 kDa band were found towards the C-terminus ofthe protein. It appears that the 25 and 45 kDa bands on the SDS-PAGE arefragments of the full-length hypothetical protein. Comparison of thehypothetical protein sequence with the GenBank sequence database byusing the BLAST program revealed a number of similar proteins frommouse, rat and bovine with unknown functions, and a PLB fromDictyostelium discoideum (accession number Q8MWQ0). The amino acidsequence of the hypothetical protein has 32% identity with that of PLBfrom Dictyostelium discoideum.

Enzyme assays. To determine a possible deacylation activity of theputative PLB, freshly purified protein and materials from differentsteps of purification were incubated with either of several differentsubstrates including didecanoyl-phosphatidylcholine (didecanoyl-PC),dipalmitoylphosphatidylcholine (dipalmitoyl-PC), phosphatidylinositol(PI), dipalmitoylphosphatidylethanolamine (PE) and1-palmitoyl-2-hydroxylphosphatidylcholine (Lyso-PC). No activity wasdetected except for the acid extracts of granules. However, the purifiedprotein stored at 4° C. for some period of time removed fatty acid fromdidecanoyl-PC, and the activity increased by storage time. In additionto phosphatidylcholine deacylation, the enzyme also showed deacylationactivity on PI, PE and Lyso-PC. To investigate if a change in molecularweight was associated with the appearance of the deacylation activity,the purified protein stored at 4° C. for 16 weeks was analysed bySDS-PAGE. In addition to the major bands at 25 and 45 kDa, thereappeared minor bands at molecular weights of around 21 and 41-44 kDawhich partly shifted from the major bands, coinciding with theappearance of a significant deacylation activity. The more shifted fromthe major bands to the minor bands the more enzyme activity appeared.The enzyme is active at a broad pH range with an optimum of 7.4, whendidecanoyl-PC was used as substrate and incubated at 37° C. From theHanes plots a km of 1.1 mM and a V_(max) value of 21.4 nM/min/mg werecalculated when the protein (stored for 15 weeks) was used. It isobvious that the native purified protein needs molecular processing toacquire its activity. To investigate if activating factors are presentin granules of neutrophils, the partly activated protein (0.5 μg, storedfor 19 weeks) was pre-incubated with released materials (0.5 μg) inducedby PMA from neutrophils for 15 min at room temperature and 37° C. beforeincubation with substrate mixture. The activity was increased about 10%by pre-incubation at room temperature and 30% by pre-incubation at 37°C.

Having known that the enzyme can remove fatty acid from the sn-1position of 1-palmitoyl-2-hydroxyl-PC (Lyso-PC), it was incubated withlabeled phosphatidylcholine, 1-palmitoyl-2-[1-¹⁴C]palmitoyl-PC (GMHealth care, Uppsala). The enzyme also removed fatty acid from the sn-2position. Based on these results we conclude that we are dealing with anovel human PLB.

Antibody production and cellular localization of the novel PLB inneutrophils. By the time of immunization, the 25 kDa molecule was notidentified as part of the hypothetical protein (FLJ22662), therefore,the 25 and 45 kDa molecules were separated by preparativeelectrophoresis and the antigens were separately injected to differentchickens. The chicken given the 25 kDa molecule had no response to theantigen, while the chicken given the 45 kDa molecule had producedspecific antibodies and reacted with the 45 kDa molecule as seen on animmunoblot. To investigate the origin of the protein in humangranulocytes, neutrophil and eosinophil post nuclear supernatants wereprepared and the proteins were separated on SDS-PAGE, followed byimmunoblotting using the chicken anti-45 kDa antibodies. No band wasdetected in the post nuclear supernatant of eosinophils, but a band at amolecular weight of 45 kDa was detected in the post nuclear supernatantof neutrophils, indicating the neutrophil origin of the protein.

Discussion

This study has shown the identification, purification andcharacterization of a novel protein from acid extracts of granules ofneutrophil granulocytes of healthy blood donors by means of a simplethree-column procedure. The protein was identified as a novel PLBcontained in the secretory granules of human neutrophils. The discoverysheds new light on the role of the neutrophil in inflammation, sincethis enzyme may not only have the capacity to kill bacteria but alsohave the capacity to generate lipid mediators of inflammation in localneutrophil involving processes.

PLBs are enzymes that can remove both the sn-1 and sn-2 fatty acids ofglycerophospholipids, and thus displays both phospholipase A₂ andlysophospholipase activities. Several PLBs have been identified inbacteria (Farn et al., cited above), fungi (Ghannoum et al., citedabove), Dictyostelium discoideum (Ferber et al., cited above), inmammalian cells (Gassama-Diagne et al., cited above; Tojo et al., citedabove; Boll et al., cyed above; and Maury at al., cited above) and inbee and snake venoms. Genes coding for these PLBs were cloned and threedistinct gene families have been identified from bacteria (Farn et al.,cited above), fungi (Ghannoum et al., cited above) and mammals (Boll etal., cited above; Maury at al., cited above; Delagebeaudeuf et al., J.Biol. Chem. 273, 13407-13414 (1998); and Takemori et al., J. Biol. Chem.273, 2222-2231 (1998)). However, the gene (F1122662) is not related toany of these gene families and the coded protein lacks the typicallipase motif (Schrag et al., cited above) found in lipases andphospholipases towards the C-terminus, suggesting that the putative PLBis a member of a new gene family of PLB as described for DictyosteliumPLB (Morgan et al., Biochem. J. 382, 441-449 (2004)). We suggest thatthis novel PLB should be named Human Phospholipase B type II (HPLB-II)to distinguish this gene product from the above mentioned PLBs. SinceHPLB-II also displayed PLA₂ activity, it is fair to presume that it maybe involved in diverse biological processes, such as arachidonic acidmetabolism (Leslie C. C., J. Biol. Chem. 272, 16709-16712 (1997)),atherosclerosis (Webb et al., Arterioscler. Thromb. Vasc. Biol. 23,263-268 (2003)) and antibacterial defence (Buckland et al., Biochim.Biophys. Acta. 1488, 71-82 (2000); and (Koduri et al., J. Biol. Chem.277, 5849-5857 (2002)).

It was obvious that HPLB-II needs molecular processing to acquire itsdeacylation activity. Similar observation was made in guinea pigintestinal PLB, which is produced as a pro-enzyme activated uponshifting the molecular weight from 170 kDa to 140 kDa by trypsintreatment (Delagebeaudeuf et al., (1998) cited above). Materials fromdifferent steps of purification (step 2-4) showed no deacylationactivity except for the acid extracts of granules. The activity seen inthe acid extracts of granules was not likely due to PLA₂s present inneutrophil primary and secondary granules (Victor et al., Febs Lett.136, 298-300 (1981); and Degousee et al., J. Biol. Chem. 277, 5061-5073(2002)), because these are Ca²⁺-dependent enzyme and Ca²⁺ was not addedto our incubation mixture. Therefore, there might be factors in thegranules that lead to activation of the HPLB-II. This was confirmed bythe pre-incubation of the partly activated enzyme with releasedmaterials from neutrophils. This pre-incubation further activated theenzyme. The mechanism, however, by which HPLB-II gains activity, remainsto be investigated. Characterization of substrate specificity indicatesthat HPLB-II is not limited to hydrolyzing phosphatidyl-PC, as PI and PEalso serve as substrates. The enzyme is active at a broad pH range withan optimum of 7.4, suggesting an extracellular deacylation role. Theimmunoblotting was chosen to determine the localization of HPLB-II inhuman granulocytes, because the antigen used for immunization was in adenatured form after separation by preparative electrophoresis.Therefore, the antibodies raised in this study are suitable to detectthe denatured form of the molecule. The immunoblotting indicated aneutrophil origin of HPLB-II. However, the immunoblotting failed to showa band at a molecular weight of 63 kDa (a molecular weight of fulllength hypothetical protein FLJ22662). The explanation for this could bethat the full length protein of 63 kDa is cleaved by proteases presentin the preparation of the post nuclear supernatant. However, we findthis unlikely, since a protease inhibitor cocktail was included in thepreparation. Another explanation is that the full length protein alreadyhas been processed into fragments of 25 and 45 kDa duringgranulopoiesis.

The fragments 25 and 45 kDa were seen on SDS-PAGE under both reducingand non-reducing conditions. However, the two molecules were notseparated by chromatographic procedures applied in this study includingchromatofocusing and reversed phase chromatography. These findings andthe apparent molecular weight of 130 kDa by gel filtration suggest thatthe protein in fact is an oligomeric protein comprising at least two 25kDa and two 45 kDa molecules associated non-covalently.

TABLE 1 Peptide mass fingerprint of the purified protein Residues no inNo m/z FLJ22662 25 kDa subunit 1 1129.62 47-57 2 1160.62 52-61 3 1144.6275-84 4 895.41 134-140 5 832.42 141-146 6 1230.57 151-159 7 810.37154-159 8 1821.91 160-176 45 kDa subunit 1 2685.34 233-255 2 1819.85259-273 3 1468.80 313-324 4 1921.89 345-360 5 2715.36 371-393 6 1750.85394-407 7 1622.75 395-407 8 1381.69 421-432 9 1251.48 466-475 10 2972.53493-520 11 2595.32 527-548 The amino acid sequences/fragments shown inbold were determined by MS/MS

Quantitation of the Innovative Protein in Various Biological Fluids.

A double antibody radioimmunoassay (RIA) was developed for themeasurement of HPLB-II. HPLB-II was labeled with ¹²⁵I by thechloramine-T method. Free ¹²⁵I was separated from labeled protein by gelfiltration on a Sephadex G-25 column. Before RIA incubation, the labeledantigen was diluted in assay buffer (50 mM sodium phosphate, pH 7.4,containing 80 mM NaCl, 10 Mm Na-EDTA, 0.2% Bovine serum albumin, 0.02%NaN₃ 0.2% CTAB and 0.5% Tween 20) to 38000±2000 cpm/100 μl (tracer). A50 μl solution of either sample or standard (2-128 μl/l) wassequentially mixed with 50 μl of labeled HPLB-II (tracer), 50 μl ofspecific antibodies raised in rabbit (diluted 1:60000 in assay buffer)and incubated for 3 h at room temperature. Thereafter, 0.5 ml ofdecanting suspension containing Sephadex anti-rabbit IgG raised in sheepwere added and the incubation continued for 1 h at 4° C.HPLB-II-antibody complexes bound on Sephadex anti-rabbit IgG wereseparated and pelleted by centrifugation at 18° C. for 15 mM at 4000rpm. After decantation, the radioactivity was measured in a gammacounter.

Results: A standard curve indicating a possible measuring range 1-100μg/L of PLB-II (four subunits) with a limit of detection of 1 μg/L.PLB-II in normal serum was undetectable whereas raised levels were foundin serum obtained from patients with acute bacterial infections (3-9μg/L). Highly raised levels were also found in lung lavage fluids ofpatients with chronic obstructive pulmonary disease (1-21 μg/L) andintestinal fluid of patients with inflammatory bowel disease (22-52μg/L).

Certain innovative aspects of the invention are defined in more detailin the appending claims. Although the present invention and itsadvantages have been described in detail, it should be understood thatvarious changes, substitutions and alterations can be made hereinwithout departing from the spirit and scope of the invention as definedby the appended claims. Moreover, the scope of the present applicationis not intended to be limited to the particular embodiments of theprocess, machine, manufacture, composition of matter, means, methods andsteps described in the specification. As one of ordinary skill in theart will readily appreciate from the disclosure of the presentinvention, processes, machines, manufacture, compositions of matter,means, methods, or steps, presently existing or later to be developedthat perform substantially the same function or achieve substantiallythe same result as the corresponding embodiments described herein may beutilized according to the present invention. Accordingly, the appendedclaims are intended to include within their scope such processes,machines, manufacture, compositions of matter, means, methods, or steps.

The invention claimed is:
 1. A method for determining the occurrence orlevel of a mammalian protein in a sample, wherein the mammalian proteinis pro-phospholipase B (PLB-II) and comprises at least one SU2 subunitcomprising SEQ ID NO: 3, the SU2 subunit having a molecular weightwithin the range of 30-60 kDa, the method comprising: contacting thesample with a phospholipase B enzymatic reactant, and determining thepresence or amount of consumed or remaining enzymatic reactant aftersuch contact.
 2. The method of claim 1, for diagnosing a diseasecondition associated with an abnormal level of the mammalian protein ina mammalian patient, wherein the method further comprises comparing thedetermined amount of enzymatic reactant with an amount of enzymaticreactant representative of healthy individuals of the same species,wherein a determined amount that deviates from the amount representativeof healthy individuals of the same species is an indication that saidpatient suffers from the disease condition.
 3. The method of claim 2,wherein the disease condition is i) an inflammatorydisease/inflammation, ii) a microbial infection, iii) a cardiovasculardisease, or iv) a lipid disorder.
 4. The method of claim 2, wherein thedisease condition is selected from the group consisting of bacterialinfection, chronic obstructive pulmonary disease, and inflammatory boweldisease.
 5. The method of claim 2, wherein the disease condition is alipid disorder involving neutrophil granulocytes and/ormonocytes/macrophages.
 6. The method of claim 1, wherein the mammalianprotein comprises an SU2 subunit and is devoid of an SU1 subunitcomprising SEQ ID NO: 2 and having a molecular weight within the rangeof 15-35 kDa.
 7. The method of claim 1, wherein the mammalian proteincomprises at least one SU1 subunit comprising SEQ ID NO: 2 and having amolecular weight within the range of 15-35 kDa, wherein the SU1 subunitand the SU2 subunit are derived from the same mammalian species.
 8. Themethod of claim 7, wherein the mammalian protein has a molecular weightof up to 240 kDa.
 9. The method of claim 7, wherein the mammalianprotein comprises two SU1 subunits and two SU2 subunits.
 10. The methodof claim 9, wherein the SU1 subunits are of 25 kDa molecular weight andthe SU2 subunits are of 45 kDa molecular weight.
 11. The method of claim7, wherein the SU1 subunits are of 25 kDa molecular weight and the SU2subunits are of 45 kDa molecular weight.
 12. The method of claim 1,wherein the mammalian protein is devoid of a lipase consensus motif offive amino acid residues having glycine at both end positions and serinein the middle position.
 13. The method of claim 1, wherein the mammalianprotein is labelled with an analytically detectable tag, is immobilizedto a solid phase, or is equipped with an immobilizing tag that rendersthe mammalian protein immobilizable to a solid phase.
 14. A method fordetermining the capability of a compound to transform or to inhibit thetransformation of a selected mammalian protein in pro-phospholipase Bform to an enzyme active phospholipase B form, comprising the steps of:(i) selecting a mammalian protein comprising at least one SU2 subunitcomprising SEQ ID NO: 3, the SU2 subunit having a molecular weightwithin the range of 30-60 kDa, which has been determined aspro-phospholipase B; (ii) bringing the selected mammalian protein, asubstrate for phospholipase B, and the compound in contact with eachother under activating conditions, (iii) measuring the conversion of thesubstrate as a function of time, and (iv) determining from saidconversion the capability of the compound to activate the protein tophospholipase B.
 15. The method of claim 14, wherein the mammalianprotein comprises an SU2 subunit and is devoid of an SU1 subunitcomprising SEQ ID NO: 2 and having a molecular weight within the rangeof 15-35 kDa.
 16. The method of claim 14, wherein the mammalian proteincomprises at least one SU1 subunit comprising SEQ ID NO: 2 and having amolecular weight within the range of 15-35 kDa, wherein the SU1 subunitand the SU2 subunit are derived from the same mammalian species.
 17. Themethod of claim 16, wherein the mammalian protein comprises two SU1subunits and two SU2 subunits.
 18. The method of claim 17, wherein theSU1 subunits are of 25 kDa molecular weight and the SU2 subunits are of45 kDa molecular weight.
 19. A method for determining the capability ofa compound to enhance or inhibit the enzymatic activity of a selectedprotein having phospholipase B enzyme activity, comprising the steps of:(i) selecting a protein comprising an activated form of a mammalianprotein comprising at least one SU2 subunit comprising SEQ ID NO: 3, theSU2 subunit having a molecular weight within the range of 30-60 kDa,wherein the activated form has been determined as having phospholipase Benzyme activity; (ii) bringing the selected protein, a substrate forphospholipase B, and the compound in contact with each other underactivating conditions, (iii) measuring conversion of the substrate as afunction of time, and (iv) determining from said conversion thecapability of the compound to enhance or inhibit the phospholipase Benzyme activity.
 20. The method of claim 19, wherein the selectedprotein has a molecular weight that is at least 0.5 kDa lower thannon-activated mammalian protein.